Stable commercial preparations of thermophilic polymerases that retain during storage and in reaction mixtures the same level of the enzymatic activity are necessary for their use in many biochemical processes or diagnostics. Thermophilic polymerases can be native (isolated from naturally found thermophilic microorganisms) or recombinant (isolated from the bacterial host harbouring cloned gene encoding said thermophilic polymerase). Due to their origin these enzymes are stable to denaturation by heat. However, even highly thermostable polymerases may be inactivated by chemical agents, proteases, or unfavourable environmental conditions during storage.
Noteworthy, many uses of thermostable enzymes often comprise reaction steps in denaturing conditions, such as highly elevated temperatures, aqueous environments with sub-optimal concentrations of cofactors and substrates, and a pH that is suboptimal for maximum enzyme stability. For example, amplification of nucleic acids involves thermal cycling of a reaction mixture containing a nucleic acid polymerase to generate an amplified target nucleic acid product. An example of a thermal cycling process is Polymerase Chain Reaction (PCR), in which the reaction mixture is subjected to oligonucleotide denaturing, primer annealing, and primer extension reaction temperatures. Thermostable DNA polymerases are generally used to amplify target DNA sequences in said thermal cycling reactions.
Numerous stabilization techniques are known. These techniques include chemical modification of the enzyme, genetic engineering of the enzyme and the addition of stabilizing additives, such as detergents. Detergents are surface active compounds that are conventionally considered to stabilize the interface between the hydrophobic enzyme surface and the hydrophilic liquid environment in which they are contained. For example, U.S. Pat. No. 6,127,155 B1 (included herein as reference) discloses that non-ionic polymeric detergents, such as polyethoxylated sorbitan monolaurate (Tween 20) and ethoxylated alkyl phenol (NP-40) contained in the storage buffer stabilize Taq DNA polymerase. Also, U.S. Pat. No. 6,242,235 B1 discloses polymerase stabilization by polyethoxylated amine surfactants. Also disclosed therein are cationic surfactants for the stabilization of polymerases. Similar compounds have been proposed as polymerase stabilising agents in US patent application US2003/0134292A1.
U.S. Pat. No. 6,242,235 B1 discloses polymerase stabilization by polyethoxylated amine surfactants, while WIPO PCT patent application WO/2008/013885A2 discloses the use of zwitterionic detergents and non-detergent surfactants for the storage and uses of DNA polymerases. EPO patent application EP1970440 A1 discloses stabilization of polymerases in aqueous solutions by ionic, particularly zwitterionic detergents in the presence of inert proteins, such as BSA. WIPO patent application WO/2008/077017A2 discloses compositions, methods, and kits comprising thermostable DNA polymerase and an anionic detergent or a zwitterionic detergent.
All above cited stabilization methods have certain drawbacks, including (i) high denaturing effect, (ii) a positive or negative charge, (iii) a low efficiency in disrupting polymerase protein aggregation and (iv) an often difficult removal of the detergent after the performance of the reaction, especially in cases when presence of detergent is undesirable in downstream applications (e.g. use in microarrays). For example, PCR products used for DHPLC (denaturing high pressure liquid chromatography) analysis are recommended to be free of detergents. Detergents may also cause foaming when PCR products are spotted on microarray slides (www.finnzymes.com/pcr/phusion_products.html).
Thus, there is a need for methods and compositions comprising stabilized thermophilic nucleic acids polymerases, which have low denaturing effect, high efficiency in disrupting aggregation and which need no detergent in case of interference in the further processing of polymerization reaction products.